Gill-associated nidovirus of Penaeus monodon prawns transcribes 3′-coterminal subgenomic mRNAs that do not possess 5′-leader sequences

Cowley, Jeff A.; Dimmock, Christine M.; Walker, Peter

doi:10.1099/0022-1317-83-4-927

Cited by 63 publications

(97 citation statements)

References 41 publications

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“…1A). For two other nidovirus subgroups, roniviruses and toroviruses (with the exception of sg mRNA2 of the latter), sg mRNAs without a common 5Ј leader sequence have been described (3,32,45).For sg RNAs of coronaviruses and arteriviruses, and also the largest sg RNA of toroviruses (45), the fusion of the sg RNA body to the common leader sequence has been postulated to involve the discontinuous extension of minus-strand RNA synthesis, which presumably yields sg minus-strand templates that are used to produce the sg plus strands (31). After attenuation of RNA synthesis, the nascent minus strand is thought to be translocated to the 5Ј-proximal region of the genomic template, where RNA synthesis is resumed to add the complement of the genomic leader sequence, thus completing the minusstrand template for sg mRNA synthesis (Fig.…”

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Discontinuous Subgenomic RNA Synthesis in Arteriviruses Is Guided by an RNA Hairpin Structure Located in the Genomic Leader Region

Born

Posthuma

Gultyaev

et al. 2005

J Virol

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Nidoviruses produce an extensive 3-coterminal nested set of subgenomic (sg) mRNAs, which are used to express structural proteins and sometimes accessory proteins. In arteriviruses and coronaviruses, these mRNAs contain a common 5 leader sequence, derived from the genomic 5 end. The joining of the leader sequence to different segments derived from the 3-proximal part of the genome (mRNA bodies) presumably involves a unique mechanism of discontinuous minus-strand RNA synthesis in which base pairing between sense and antisense transcription-regulating sequences (TRSs) plays an essential role. The leader TRS is present in the loop of a hairpin structure that functions in sg mRNA synthesis. In this study, the minimal sequences in the 5-proximal region of the Equine arteritis virus genome that are required for sg RNA synthesis were delimited through mutagenesis. A full-length cDNA clone was engineered in which this domain was duplicated, allowing us to make mutations and monitor their effects on sg RNA synthesis without seriously affecting genome replication and translation. The leader TRS present in the duplicated sequence was used and yielded novel sg mRNAs with significantly extended leaders. Our combined findings suggest that the leader TRS hairpin (LTH) and its immediate flanking sequences are essential for efficient sg RNA synthesis and form an independent functional entity that could be moved 300 nucleotides downstream of its original position in the genome. We hypothesize that a conformational switch in the LTH region regulates the role of the 5-proximal region of the arterivirus genome in subgenomic RNA synthesis.Many eukaryotic plus-strand RNA (ϩRNA) viruses generate subgenomic (sg) mRNAs as a strategy to regulate the expression of one or several viral proteins (21). In addition to internal initiation of translation, sg mRNA synthesis provides a mechanism to express genes positioned downstream of the 5Ј-proximal gene in polycistronic genomes of ϩRNA viruses of eukaryotes. Different mechanisms are employed by ϩRNA viruses to achieve the transcription of sg mRNAs (47). The generation of an extensive 3Ј-coterminal nested set of sg mRNAs to express structural and/or accessory proteins is a common property of viruses belonging to the order Nidovirales (Coronaviridae, Arteriviridae, and Roniviridae) (6, 34). In the case of arteriviruses and coronaviruses, all transcripts contain a common 5Ј sequence element, the so-called "leader," which is colinear with the 5Ј-proximal part of the genome and is fused to different regions derived from the 3Ј-proximal third of the genome, which are termed "mRNA bodies" (Fig. 1A). For two other nidovirus subgroups, roniviruses and toroviruses (with the exception of sg mRNA2 of the latter), sg mRNAs without a common 5Ј leader sequence have been described (3,32,45).For sg RNAs of coronaviruses and arteriviruses, and also the largest sg RNA of toroviruses (45), the fusion of the sg RNA body to the common leader sequence has been postulated to involve the discontinuous extension of minu...

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Discontinuous Subgenomic RNA Synthesis in Arteriviruses Is Guided by an RNA Hairpin Structure Located in the Genomic Leader Region

Born

Posthuma

Gultyaev

et al. 2005

J Virol

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“…Although qRT-PCR can accurately quantify the GAV RNA template number, it should be noted that this does not directly relate to the number of infectious virus particles. GAV dsRNA replicative intermediates have been detected in total RNA isolated from infected cells (Cowley et al 2002b), and cDNA generated to both (+) and (-) sense RNA strands will be amplified in the PCR. Moreover, non-encapsidated, filamentous nucleocapsid precursors that contain GAV genomic RNA, but which are likely to be far less infectious than enveloped particles, are usually observed in far greater numbers in infected cells than are mature virions (Spann et al 1997).…”

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Quantitative real-time RT-PCR demonstrates that handling stress can lead to rapid increases of gill-associated virus (GAV) infection levels in Penaeus monodon

Vega¹,

Degnan²,

Hall³

et al. 2004

Dis. Aquat. Org.

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Gill-associated virus (GAV) of the black tiger prawn Penaeus monodon has been implicated as a cause of periodic production losses in Australia since 1996. We report here the development of a real-time quantitative RT-PCR (qRT-PCR) for GAV. A dilution series of in vitro transcribed RNA was used to determine the sensitivity limit of the qRT-PCR and as a standard for GAV quantification. A linear relationship between cycle threshold (Ct) values and input RNA was obtained over a wide concentration range between 4.86 × 10 9 and 0.5 template copies per reaction, the latter being the test detection limit. The qRT-PCR was used to follow the progression of GAV levels in a group of 15 adult male P. monodon with chronic GAV infections that were super-infected by intramuscular injection of an inoculum containing high levels of GAV. By Day 9 post-injection, cumulative mortalities reached 100% (15/15) in the GAV-injected prawns and 40% (2/5) in placebo-injected prawns. Spermatophores were collected at the beginning, and together with other tissues, at the end of the trial. Prawns were also bled at regular intervals to collect circulating haemocytes. The qRT-PCR revealed that GAV loads increased significantly in haemocytes collected from both the control and super-infected prawns (p = 0.010). This increase was significantly higher in the super-infected prawns (p = 0.047). The rapid increase in GAV levels in super-infected P. monodon was expected. However, the increase in the control prawns was not, and indicates that repetitive bleeding and handling stress can stimulate GAV proliferation in chronically infected P. monodon. KEY WORDS: Penaeus monodon · Gill-associated virus · Quantitative RT-PCR (qRT-PCR)Resale or republication not permitted without written consent of the publisher Dis Aquat Org 59: 195-203, 2004 2003, Tang et al. 2002, Callinan et al. 2003. Sequence comparisons of viruses from many healthy and moribund P. monodon indicate that they are genetically inseparable and are now regarded as the same virus (Cowley et al. 2000 and unpubl. data).GAV is a positive-strand ssRNA nidovirus closely related to the yellow head virus (YHV) from Thailand in genome sequence and organisation (Cowley et al. 1999. GAV and YHV have recently been classified in a new genus (Okavirus) and family (Roniviridae) within the Nidovirales (Mayo 2002). Both viruses have rod-shaped enveloped particles and cause similar histopathology and cytopathology (Spann et al. 1995, 1997, Tang & Lightner 1999, Tang et al. 2002. GAV diagnosis based on clinical signs and histopathology is not particularly specific as other viruses can cause similar effects. More precise diagnostic methods include transmission electron microscopy (TEM), in situ hybridisation (ISH) (Tang et al. 2002) and RT-nested PCR (Cowley et al. 2000).There is no detailed quantitative data directly linking GAV infection levels to the manifestation of disease symptoms. However, histological and ISH data on healthy and moribund Penaeus monodon and P. esculentus experimentally infec...

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“…The large sizes of the replicase-associated genes and interdomain areas, in addition to the large number of sgRNAs, suggest similarity to the Nidovirales. However, the lack of discontinuous mRNAs with a common leader (21), characteristic of the Arteriviruses and Coronaviruses of the Nidovirales (29,52), suggest greater similarity to the alpha-like viruses, although most of the sgRNAs of the toroviruses and okaviruses of the Nidovirales are contiguous with the 3Ј end of the genome like CTV (12,53,59). RNA-dependent RNA polymerase (RdRp) phylogeny, however, clearly places the closteroviruses into the alphavirus-like supergroup (27).…”

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“…Most of the small RNA viruses of plants have guanylates as 5Ј termini of the genomic and sgRNAs (8,13,16,18,23,25,26,48,55,56,60,61,63,65). Adenylate, however, is a common 5Ј terminus of RNAs for animal alpha-like viruses and members of the Nidovirales (12,31,38,53,59) but infrequent for plant viruses (30,43,67). Uridylate and cytidylate as 5Ј termini are uncommon but have been also reported for rubella virus and Oat chlorotic stunt virus sgRNAs, respectively (7,42).…”

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Effects of Modification of the Transcription Initiation Site Context on Citrus Tristeza Virus Subgenomic RNA Synthesis

Ayllón

Gowda

Satyanarayana

et al. 2003

J Virol

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Citrus tristeza virus (CTV), a member of the Closteroviridae, has a positive-sense RNA genome of about 20 kb organized into 12 open reading frames (ORFs). The last 10 ORFs are expressed through 3-coterminal subgenomic RNAs (sgRNAs) regulated in both amounts and timing. Additionally, relatively large amounts of complementary sgRNAs are produced. We have been unable to determine whether these sgRNAs are produced by internal promotion from the full-length template minus strand or by transcription from the minus-stranded sgRNAs. Understanding the regulation of 10 sgRNAs is a conceptual challenge. In analyzing commonalities of a replicase complex in producing so many sgRNAs, we examined initiating nucleotides of the sgRNAs. We mapped the 5 termini of intermediate-(CP and p13) and low-(p18) produced sgRNAs that, like the two highly abundant sgRNAs (p20 and p23) previously mapped, all initiate with an adenylate. We then examined modifications of the initiation site, which has been shown to be useful in defining mechanisms of sgRNA synthesis. Surprisingly, mutation of the initiating nucleotide of the CTV sgRNAs did not prevent sgRNA accumulation. Based on our results, the CTV replication complex appears to initiate sgRNA synthesis with purines, preferably with adenylates, and is able to initiate synthesis using a nucleotide a few positions 5 or 3 of the native initiation nucleotide. Furthermore, the context of the initiation site appears to be a regulatory mechanism for levels of sgRNA production. These data do not support either of the established mechanisms for synthesis of sgRNAs, suggesting that CTV sgRNA production utilizes a different mechanism.

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Gill-associated nidovirus of Penaeus monodon prawns transcribes 3′-coterminal subgenomic mRNAs that do not possess 5′-leader sequences

Cited by 63 publications

References 41 publications

Discontinuous Subgenomic RNA Synthesis in Arteriviruses Is Guided by an RNA Hairpin Structure Located in the Genomic Leader Region

Discontinuous Subgenomic RNA Synthesis in Arteriviruses Is Guided by an RNA Hairpin Structure Located in the Genomic Leader Region

Quantitative real-time RT-PCR demonstrates that handling stress can lead to rapid increases of gill-associated virus (GAV) infection levels in Penaeus monodon

Effects of Modification of the Transcription Initiation Site Context on Citrus Tristeza Virus Subgenomic RNA Synthesis

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